Axonal Surface Research Articles

Expression and in vitro function of beta 1-integrin laminin receptors in the developing avian ciliary ganglion [published erratum appears in J Neurosci 1996 Nov 1;16(21):7097-8

In chick development, ciliary ganglion (CG) neurons go through a period of axon extension from approximately embryonic day (E)4 to E8, followed by a period of synaptogenesis and neuronal cell death. By examining the immunohistochemical localization of laminin, in conjunction with Dil labeling of the ciliary nerve projection, we have determined that the pathway taken by these neurons is rich in laminin expression. Therefore, laminins are good candidate molecules for mediating outgrowth of these neurons in vivo. In vitro, the ability of CG neurons to extend neurites on laminin-1 (EHS laminin, alpha 1 beta 1 gamma 1) is maximal up to E8, then declines dramatically. CG neuron outgrowth on laminin-1 requires the activity of beta 1-class integrins. We have used subunit-specific antibodies to determine which of the five beta 1-containing heterodimers known to be laminin receptors (alpha 1 beta 1, alpha 2 beta 1, alpha 6 beta 1, alpha 7 beta 1) are expressed, and which mediate neurite outgrowth. While we could not detect expression of alpha 2 or alpha 7, we have found that alpha 1, alpha 3 beta 1, and alpha 6 beta 1 are expressed on the surface of ciliary ganglion neuron cell bodies and axons, both in vitro and in vivo. Furthermore, antibodies against alpha 3 and alpha 6, but not alpha 1, interfered with CG neurite outgrowth on laminin-1 in vitro. Taken together, these data suggest that interactions of cell surface alpha 3 beta 1 and alpha 6 beta 1 integrins with laminin-1 are likely to mediate growth of CG neurons during pathfinding in vivo.

Preferential addition of newly synthesized membrane protein at axonal growth cones.

The addition of plasma membrane proteins to a growing axon could occur by preferential insertion at the tip (the growth cone), by uniform insertion along the axon, or by insertion at the cell body and bulk flow along the axon. To differentiate between these possibilities we used a defective herpesvirus vector to express an exogenous protein, the lymphocyte transmembrane protein CD8 alpha, in cultured rat hippocampal neurons. The newly synthesized protein first appeared on the axonal surface almost exclusively at the growth cone. Preferential addition at the growth cone was also observed in minor processes (immature dendrites), but not in mature dendrites. Over several hours, CD8 alpha reached a uniform distribution over the entire neuronal surface, presumably by diffusion within the membrane and possibly endocytic recycling. As well as providing materials for axonal growth, the selective addition of membrane vesicles at the growth cone may contribute to the polarized distribution of axonal surface molecules.

Myelinated axon number in the optic nerve is unaffected by Alzheimer's disease.

Visual symptoms are a common but not invariable feature of Alzheimer's disease (AD) and such symptoms appear to become more pronounced as the severity of the dementia increases. Pathology in both the pregeniculate and cortical parts of the visual system has been suggested to underlie the visual deficits in AD. In order to investigate the former possibility, the effect of AD on the optic nerve was investigated. Intraorbital segments of optic nerve were taken at autopsy from nine patients with AD and seven patients with no history of psychiatric or neurological disease and no abnormal neuropathology. All patients had functional vision before death and appeared free of retinal, optic nerve, or microvascular disease. The optic nerves were processed into resin, semi-thin sections cut perpendicular to the long axis of each optic nerve, and stained with paraphenylenediamine. The sections were then investigated using an image analysis system and standard morphometric techniques. There was no significant difference in the mean cross sectional neural area of AD compared with control optic nerves. Neither were there any significant differences between myelinated axon surface density, total axon number, or mean cross sectional axon area in AD compared with control optic nerves. These results indicate that optic nerve degeneration is not a feature of AD and suggest that the visual deficits in the disease result from cortical dysfunction. This view is supported by the fact that visuospatial dysfunction appears to be the most common visual problem in AD.

Semi-automatic data acquisition for quantitative neuroanatomy. MicroTrace — computer programme for recording of the spatial distribution of neuronal populations

We present a computer programme, MicroTrace, designed for user-guided digitisation of objects in biological sections. The programme is optimised for recording the spatial distribution of neuronal structures, such as large populations of tracer-labelled cell bodies or axonal plexuses, regional borders, and surfaces. System requirements are a PC running Microsoft Windows, a microscope equipped with stepping motors, and a drawing tube. A computer generated drawing area, surrounded by menus and icons, is projected into the microscope field of view via the drawing tube. Different ‘object’ icons are assigned to individual object categories (cell types, surfaces, etc.). Digitisation is performed by pointing the cursor at objects in the section. Computer graphical symbols are superimposed on the digitised objects. All object categories are digitised, before moving the stage to other fields of view by manipulating the joystick or scroll bars. Movement of the microscope stage is accompanied by a translation of the graphical image, so that continous feedback on the progress of the digitisation is provided. MicroTrace can readily be adapted to the specific needs of the user. We show its use in different experimental neuroanatomical techniques. Two-dimensional images and three-dimensional reconstructions of neuronal distribution and surfaces are demonstrated.

Commissural fibers may guide cholinergic neuronal migration in developing rat cervical spinal cord.

The present investigation examines the role of intercellular relationships in the guidance of neuronal migration in embryonic rat cervical spinal cord. A "U-shaped" group of cholinergic neurons, was first detected on embryonic days (E) 15.5-16 surrounding the ventral proliferative zone. At these stages, no cholinergic cells were observed in the dorsal spinal cord, but by E17, many of the "U-shaped" group of cholinergic cells appeared to have translocated dorsally, to become the cholinergic dorsal horn cells seen in older animals. Between E16 and E17, these choline acetyltransferase (ChAT)-immunoreactive cells displayed primitive processes oriented dorsoventrally, suggesting migration along that axis. Two early forming substrates present in embryonic spinal cord have been implicated in the guidance of other populations of migrating neurons: glial cells organized in radial arrays and commissural axons aligned along the dorsoventral axis. Involvement of the commissural fibers with cholinergic cell migration seems more likely because the fibers and the translocation pathway have similar orientations. In double-labeling immunocytochemical studies of E15.5-17 spinal cord, some immature ChAT-containing neurons were directly adjacent to commissural fibers, as identified by SNAP/TAG-1 immunoreactivity. The temporal and spatial coincidence of developing cholinergic neurons and commissural axons is consistent with the hypothesis that these neurons could use commissural fibers as migratory guides. In addition, conventional electron micrographs were examined to determine if immature neuronal profiles were physically apposed to commissural axons. Immature neurons with leading and trailing processes oriented dorsally and ventrally, respectively, were embedded within and aligned along bundles of commissural fibers or along other similarly oriented neurons. This direct apposition of immature cells to the surfaces of commissural axons and other bipolar neurons is consistent with the hypothesis that the "U-shaped" group of cholinergic neurons may use commissural axons and other cohort neurons for guidance during their dorsal migration.

Effect of transforming growth factor-beta 1 and -beta 2 on Schwann cell proliferation on neurites.

Mechanisms regulating Schwann cell proliferation during development are unclear. Schwann cell division is known to be driven by an unidentified mitogen present on the surface of axons, but it is not known whether other molecules play a role in regulating this proliferation. Transforming growth factor-beta (TGF-beta) which is found in the developing peripheral nervous system (PNS) and is mitogenic for neuron-free Schwann cells in vitro could be involved. We have investigated the effects of TGF-beta 1, TGF-beta 2 and antibodies to TGF-beta 1 and TGF-beta 2 on axon driven Schwann cell proliferation. Rat embryonic dorsal root ganglion neurons (DRG) neurons and Schwann cells from the sciatic nerve were isolated, purified and recombined in vitro. Confirming earlier reports by others, we observed that TGF-beta 1 and TGF-beta 2 added to the culture medium stimulated the proliferation of Schwann cells in the absence of neurons. However, when added to neuron-Schwann cell co-cultures, TGF beta caused a variable response ranging from no effect to moderate inhibition of Schwann cell proliferation in different experiments. A stimulation of Schwann cell proliferation by TGF beta was never observed in neuron-Schwann cell co-cultures. Antibodies to TGF-beta 1 and TGF-beta 2 did not influence axon driven Schwann cell proliferation. To further determine the role of TGF-beta in Schwann cell proliferation and myelination, we studied Schwann cell proliferation in cultures from mice in which the TGF-beta 1 gene was delected by homologous recombination. Neuron-Schwann cell cultures from wild-type, heterozygous and homozygous mice were used. No differences were observed in either Schwann cell proliferation or myelination between cultures obtained from homozygous mutants and their heterozygous and wild-type controls. These findings suggest that TGF-beta does not function as a part of the mitogenic mechanism presented by neurons to Schwann cells, but that the presence of active TGF beta in the cellular environment might regulate the degree of proliferation induced by neuronal contact.

Axon-induced mitogenesis of human Schwann cells involves heregulin and p185erbB2.

The ability of sensory axons to stimulate Schwann cell proliferation by contact was established in the 1970s. Although the mitogen responsible for this proliferation has been localized to the axon surface and biochemically characterized, it has yet to be identified. Recently a family of proteins known as heregulins (HRGs) has been isolated, characterized, and shown to interact with a number of class 1 receptor tyrosine kinases, including the erbB2, erbB3, and erbB4 gene products. These factors include glial growth factor, a Schwann cell mitogen. We have tested the effects of antibodies against components of this system (HRG beta 1 and p185erbB2) in cocultures of rat sensory neurons and human (or rat) Schwann cells to elucidate the role of these proteins in axon-induced Schwann cell proliferation. 2C4, a monoclonal antibody specific for the human p185erbB2 receptor tyrosine kinase, bound to the surface of human Schwann cells and reduced human Schwann cell incorporation of [3H]thymidine by > 90% compared with untreated controls in this coculture system. This antibody had no effect on rat Schwann cell incorporation of [3H]thymidine under similar conditions. A polyclonal antibody raised against HRG beta 1 reduced human and rat Schwann cell incorporation of [3H]thymidine by nearly 80% and up to 49%, respectively, relative to controls. These results imply that a HRG, or a HRG-like molecule, is a component of the axonal mitogen. This mitogen is presented to Schwann cells by axons and induces proliferation through an interaction that involves p185erbB2 on Schwann cells.

Aplysia cell adhesion molecules and serotonin regulate sensory cell- motor cell interactions during early stages of synapse formation in vitro

Long-term facilitation of sensorimotor synapses of Aplysia in culture by serotonin (5-HT) is accompanied by two changes: an increase in the number of sensory cell branches and varicosities contacting the major axons of the target motor cell L7, and a downregulation of Aplysia cell adhesion molecules (apCAM) from the surface of the presynaptic sensory cell. We tested the hypothesis that the two changes may be linked; the 5-HT-induced decrease of apCAM levels from sensory neurites may defasciculate sensory neurites from each other and make the surface of the motor axons a more attractive substrate for new growth and synapses. We used developing cultures to examine the relationship of neuritic branching, varicosity formation, and efficacy of the connections formed by sensory cells to levels of apCAM expression on the motor cell. We then determined the consequences of 5-HT applied during the early period of interaction between sensory and motor cells (day 1 or 2 in culture) on the pattern of sensory cell growth and synapse formation. We report that the number of sensory cell branches and varicosities, and the ability of sensory growth cones to fasciculate with L7 axons and form chemical connections correlate with the level of apCAM expression on different regions of L7. Early exposure to 5-HT increased the number of sensory cell branches and varicosities contacting newly regenerated distal neurites of L7 to levels that would normally occur when the sensory neurites interact with the major proximal axons of L7. Treatment with 5-HT also modulated the efficacy of the developing synaptic connections. The change in synapse efficacy was accompanied by an increase in the formation of new sensory varicosities and branches with pioneering growth cones extending on the major axons of L7. The results are consistent with the hypothesis that treatment with 5-HT modulates local differences in the expression of cell adhesion molecules on the surface of the interacting cells making motor neurites more attractive for sensory growth cones, thereby affecting new sensory neuritic growth and synapse formation.

Regulation of SC1/DM-GRASP during the migration of motor neurons in the chick embryo brain stem.

The hindbrain of the chick embryo contains three classes of motor neurons: somatic, visceral, and branchial motor. During development, somata of neurons in the last two classes undergo a laterally directed migration within the neuroepithelium; somata translocate towards the nerve exit points, through which motor axons are beginning to extend into the periphery. All classes of motor neuron are immunopositive for the SC1/DM-GRASP cell surface glycoprotein. We have examined the relationship between patterns of motor neuron migration, axon outgrowth, and expression of the SC1/DM-GRASP mRNA and protein, using anterograde or retrograde axonal tracing, immunohistochemistry, and in situ hybridization. We find that as motor neurons migrate laterally, SC1/DM-GRASP is down-regulated, both on neuronal somata and axonal surfaces. Within individual motor nuclei, these lateral, more mature neurons are found to possess longer axons than the young, medial cells of the population. Labelling of sensory or motor axons growing into the second branchial arch also shows that motor axons reach the muscle plate first, and that SC1/DM-GRASP is expressed on the muscle at the time growth cones arrive.

Axonal transport of herpes simplex virions to epidermal cells: evidence for a specialized mode of virus transport and assembly.

To examine the transmission of herpes simplex virus (HSV) from axon to epidermal cell, an in vitro model was constructed consisting of human fetal dorsal root ganglia cultured in the central chamber of a dual-chamber tissue culture system separated from autologous skin explants in an exterior chamber by concentric steel cylinders adhering to the substratum through silicon grease and agarose. Axons grew through the agarose viral diffusion barrier and terminated on epidermal cells in the exterior chamber. After inoculation of HSV onto dorsal root ganglia, anterograde axonal transport of glycoprotein and nucleocapsid antigen was observed by confocal microscopy to appear in exterior chamber axons within 12 h and in epidermal cells within 16 h, moving at 2-3 mm/h. Although both enveloped and unenveloped nucleocapsids were observed in the neuronal soma by transmission electron microscopy, only nucleocapsids were observed in the axons, closely associated with microtubules. Nodule formation at the surface of HSV-infected axons, becoming more dense at the axon terminus on epidermal cells, and patches of axolemmal HSV glycoprotein D expression were observed by scanning (immuno)electron microscopy, probably representing virus emerging from the axolemma. These findings strongly suggest a specialized mode of viral transport, assembly, and egress in sensory neurons: microtubule-associated intermediate-fast anterograde axonal transport of unenveloped nucleocapsids with separate transport of glycoproteins to the distal regions of the axon and assembly prior to virus emergence at the axon terminus.

Rat olfactory neurons can utilize the endogenous lectin, L-14, in a novel adhesion mechanism.

L-14 is a divalent, lactosamine-binding lectin expressed in many vertebrate tissues. In the rat nervous system, L-14 expression has been observed previously in restricted neuronal subsets within the dorsal root ganglia and spinal cord. In this study we report that L-14 is expressed by nonneuronal cells in the rat olfactory nerve. We demonstrate that L-14 binds and co-localizes with two ligands in the rat olfactory system: a beta-lactosamine-containing glycolipid, and a putative member of the laminin family. The former is expressed on the surfaces of nascent olfactory axons originating from neuron cell bodies in the olfactory epithelium. The latter is present in the extracellular matrix of the axonal path leading to synaptic targets in the olfactory bulb. In vitro, we find that recombinant L-14 promotes primary olfactory neuron adhesion to two laminin family members, and promotes intercellular adhesion. Both activities are dose-dependent, and are independent of integrin-mediated mechanisms. We have thus found that L-14 can serve two distinct adhesive functions in vitro, and propose that L-14 in vivo can promote olfactory axon fasciculation by crosslinking adjacent axons and promote axonal adhesion to the extracellular matrix.

TAG-1 can mediate homophilic binding, but neurite outgrowth on TAG-1 requires an L1-like molecule and β1 integrins

Subsets of axons in the embryonic nervous system transiently express the glycoprotein TAG-1, a member of the subfamily of immunoglobulin (Ig)-like proteins that contain both C2 class Ig and fibronectin type III domains. TAG-1 is attached to the cell surface by a glycosylphosphatidylinositol linkage and is secreted by neurons. In vitro studies have shown that substrate-bound TAG-1 promotes neurite outgrowth. We have examined the nature of axonal receptors that mediate the neurite-outgrowth promoting properties of TAG-1. Although TAG-1 can mediate homophilic binding, neurite outgrowth on a substrate of TAG-1 does not depend on the presence of TAG-1 on the axonal surface. Instead, neurite outgrowth on TAG-1 is inhibited by polyclonal antibodies directed against L1 and, independently, by polyclonal and monoclonal antibodies against beta 1-containing integrins. These results provide evidence that TAG-1 can interact with cell surfaces in both a homophilic and heterophilic manner and suggest that neurite extension on TAG-1 requires the function of both integrins and an L1-like molecule.

Membrane traffic in polarized neurons in culture.

Fetal hippocampal neurons develop axons and dendrites in culture. To study how neurons form and maintain different plasma membrane domains, hippocampal neurons were infected with RNA viruses and the distribution of the viral glycoproteins was analyzed by light and electron microscopy. Infection of hippocampal cells with vesicular stomatitis virus (VSV) and fowl plague virus (FPV) resulted in the polarized distribution of the newly synthesized viral glycoproteins. The VSV glycoprotein appeared firstly in the Golgi apparatus and then in the dendrites. In contrast, the hemagglutinin of FPV, after accumulation in the Golgi apparatus, moved to the axons. These results suggest that the mechanism of sorting of viral glycoproteins might be similar in neurons and MDCK cells, a cell line of epithelial origin. In these cells the VSV glycoprotein and the hemagglutinin of FPV distribute to the basolateral and apical membranes, respectively. Transport of viral glycoproteins to both neuronal domains was microtubule dependent. Nocodazole treatment of infected neurons inhibited the delivery of axonal and dendritic viral glycoproteins equally. To investigate if the analogy between epithelial cells and neurons extended to include an endogenous plasma membrane protein, the distribution of Thy-1, a GPI-linked protein, was analyzed. By immunofluorescence and immunoelectron microscopy, Thy-1 was found exclusively along the axonal surface. In epithelial cells GPI-anchored proteins are located apically. The existence of a barrier on the neuronal plasma membrane that would prevent intermixing of axonal and dendritic proteins was analyzed by a liposomefusion assay.(ABSTRACT TRUNCATED AT 250 WORDS)

The irregular chiasm C-roughest locus of Drosophila, which affects axonal projections and programmed cell death, encodes a novel immunoglobulin-like protein.

The axonal projection mutations irregular chiasm C of Drosophila melanogaster comap and genetically interact with the roughest locus, which is required for programmed cell death in the developing retina. We cloned the genomic region in 3C5 by transposon tagging and identified a single transcription unit that produces a major, spatially and temporally regulated mRNA species of approximately 5.0 kb. Postembryonic expression is strong in the developing optic lobe and in the eye imaginal disc. The gene encodes a transmembrane protein of 764 amino acids with five extracellular immunoglobulin-like domains and similarity to the chicken axonal surface glycoprotein DM-GRASP/SC1/BEN. Both known irreC alleles reduce the level of transcription, whereas the roughestCT mutation disrupts the intracellular domain of the protein.

Preganglionic autonomic motor neurons display normal translocation patterns in slice cultures of embryonic rat spinal cord

Phenotypic differences between somatic and autonomic motor neurons (SMNs and AMNs, respectively) may be modulated by epigenetic factors during the histogenic migrations of these cells. In order to study this problem experimentally, we have developed an in vitro, organotypic slice preparation of embryonic rat spinal cord. Our main objectives for this preparation were to determine whether in vivo patterns of motor neuronal translocations were mimicked in vitro, and, if they were, to begin to analyze such movements with experimental procedures that cannot be applied to the study of mammalian spinal cord development in vivo. Using a modification of existing organotypic slice procedures, we have shown that ChAT, an axonal surface glycoprotein and a low-molecular-weight neurofilament protein are expressed in slices cultured for up to 21 d, thus indicating that spinal neurons remained viable in vitro for relatively long periods. Most importantly, retrograde labeling and subsequent confocal microscopy have shown that the SMNs and AMNs of the slice preparations become segregated ventrodorsally into two distinct subcolumns as seen in vivo. The formation of separate AMN and SMN subcolumns appears to result from a dorsal translocation of AMNs. The fact that this cellular movement occurs in the slice preparation has allowed us to follow this phenomenon directly within the same specimen over a period of days. In addition, we have been able to observe the translocation of AMNs following the removal of their peripheral synaptic targets. The results of these experiments provide further evidence that AMNs undergo a dorsal translocation during the course of spinal cord development, and that this cellular movement may be due to an active migration. They also indicate that AMN movement is not dependent upon continual connection of these neurons with the paravertebral sympathetic ganglia.

Cell-cell adhesion by homophilic interaction of the neuronal recognition molecule axonin-1.

The axonal surface glycoprotein axonin-1, which occurs both as a glycosyl-phosphatidylinositol-anchored membrane-bound form and a secreted form, promotes neurite outgrowth and is thought to be involved in axon-guidance mechanisms in the developing nervous system. Recently, we have demonstrated that the neurite-outgrowth-promoting activity of axonin-1, presented as a substratum for cultured neurons, is mediated by a heterophilic interaction with the axonal glycoprotein neuronglia cell-adhesion molecule (Ng-CAM). Here we present evidence for homophilic (like-like) binding among axonin-1 molecules. Axonin-1 was heterologously expressed in myeloma cells. Clonal cell lines, with exposed membrane-bound axonin-1 at their surface, formed large multicellular aggregates. Incubations of transfected and parental myeloma cells, under a series of different conditions, revealed homophilic axonin-1/axonin-1 interactions across the intermembrane space as the molecular mechanism promoting stable cell-cell contacts. Using structural and functional characterisation, recombinant axonin-1 was very similar to native axonin-1, suggesting that homophilic axonin-1 interactions are also established in neurons. The capability of axonin-1 to interact with both Ng-CAM and other axonin-1 molecules might contribute to the formation of macromolecular networks at contact sites of growth cones and axons, comprising molecules of both membranes, and thus represent a mechanism for regulating neurite outgrowth and pathfinding.

Microenvironmental changes during axonal regrowth in the optic nerve of the myelin deficient rat. Immunocytochemical and ultrastructural observations

Lesion-induced regenerative sprouting of CNS axons is accompanied by reactions of the supporting glia and vascular and connective tissue which may influence the extent of regeneration. In a previous report, it was shown that after crush injury, the amyelinated optic nerve of the myelin deficient (md) mutant rat contains greater numbers of regrowing axons proximal to the site of crush than that of normally myelinated littermates. The present study was designed to compare the response of the microenvironment, i.e. glial cells and vascular and connective tissue, in md and normally myelinated optic nerves 2, 4 and 6 days after crush injury. In unoperated normal optic nerves monoclonal antibodies to the HNK-1 carbohydrate labelled astrocytic processes at the ultrastructural level whereas in unoperated md mutants HNK-1 staining was restricted to axonal surfaces. Immunoreactivity with monoclonal antibodies to stage-specific embryonic antigen-1 (SSEA-1) was confined to astrocytic surfaces in both md and wildtype animals. After axotomy of md optic nerves regrowing axons were more numerous in the proximal site of the crush and extended further into the lesion than in wildtype animals. In both md and wildtype rats regrowing axons were HNK-1-positive. In md rats strong reaction with antibodies to laminin and fibronectin was only seen in 6-day-old lesions of md rats whereas immunoreactivity was less distinct in operated littermate controls. Immunolabelling was obviously associated with blood vessels, since crush lesions in both md and wildtype rats were Schwann cell-free as assessed by electron microscopy and immunocytochemistry. In both operated md and normal littermates crush lesions contained degenerating astrocytes as well as reactive astrocytes in which the intermediate filaments of the perikarya failed to stain immunocytochemically for GFAP, vimentin, desmin, and a common determinant of intermediate filaments. In contrast, reactive astrocytes in the lesion site of normally myelinated rats expressed the SSEA-1 antigen intracytoplasmically whereas in md mutants astrocytes were completely SSEA-1-negative. Infiltration of crush lesions by macrophages was less extensive in md rats than in normal littermates. However the overall content of macrophages in the peritoneal cavity was also reduced. The present study demonstrates that (1) md optic nerves lack HNK-1-reactive astrocytes; (2) in the axotomized wildtype optic nerve impaired axonal regrowth may be associated with distinct immuno-phenotypes of the supporting glial cells, i.e. SSEA-1-positive astrocytes; (3) laminin and fibronectin seem not to be essential for improved axonal regrowth in md rats.

Ultrastructure and permeability of the Schwann cell layer surrounding the giant axon of the squid.

The ultrastructure of the Schwann cell layer surrounding the giant axon of the squid Alloteuthis subulata is described, and the permeability of extracellular compartments assessed by exposure to electron-dense tracers. Morphometric analysis is used to deduce the number, size and shape of the Schwann cells, and the routes for ion flux across the Schwann cell layer. Axons (mean diameter 233 microns) were surrounded by a 1-2 microns thick layer of Schwann cells which were approximately 1 micron thick, approximately 70 microns long and approximately 23 microns wide. There were around 62,000 Schwann cells per cm2 axon surface. The outer (abaxonal) surface of the Schwann cells was invaginated, with evidence for a covering of fine Schwann cells processes; the inner (adaxonal) surface of the Schwann cells was less folded. The percentage area occupied by mesaxonal cleft openings to the axon and to the basal lamina was 0.02% and 1.09% respectively. A system of tubules, the glial tubular system, occupied 3.9% of the Schwann cell volume, and opened to both axonal and basal lamina surfaces, with more elaborate lattice-like clusters towards the basal side of the cell. Tubule openings accounted for 0.26% of the surface area facing the axon and 0.37% of the area facing the basal lamina (where there was greater clustering of openings). The electron dense tracers horseradish peroxidase, ionic lanthanum and tannic acid filled mesaxon clefts, glial tubular system and periaxonal space. If ion flux occurred via the mesaxonal clefts, a theoretical series resistance (Rsth) of > 20 omega cm2, would be predicted, whereas if it occurred via the tubular system, the figure would be < 2 omega cm2, closer to physiological estimates. The results presented show that the glial tubular system is likely to be the major route for ion flux into and across the Schwann cell layer, and for clearance of K+ from the periaxonal space during periods of axonal stimulation. The implications for K+ homeostasis in the axonal microenvironment are discussed.

Polarized distribution of the viral glycoproteins of vesicular stomatitis, fowl plague and Semliki Forest viruses in hippocampal neurons in culture: a light and electron microscopic study

We have shown previously using immunofluorescence microscopy that upon injection of polarized hippocampal cells in culture with vesicular stomatitis virus (VSV) and fowl plague virus (FPV) the VSV glycoprotein is delivered to the plasma membrane of the dendrites and of the cell body whereas the FPV hemagglutinin is transported to the axonal surface ( Cell, 62 (1990) 63–72). In this work electron microscopy of infected rat hippocampal neurons showed that VSV progeny budded from the plasma membrane of the dendrites and the cell body. The location of the budding virions corresponded to the distribution of the VSV glycoprotein which was detected over the somatodendritic plasma membrane by immunoelectron microscopy. In contrast, no FPV formation was seen in the infected neurons although the FPV hemagglutinin was localized to the axonal surface by immunoelectron microscopy. In Semliki Forest virus (SFV) infected hippocampal cells we observed that the viral glycoproteins were exclusivelly present in the dendrites and cell body but not in axons.

Embryonic development of rat sympathetic preganglionic neurons: possible migratory substrates.

Spinal somatic and autonomic (sympathetic preganglionic) motor neurons are generated synchronously and, subsequently, migrate from the ventricular zone together to form a common primitive motor column. However, these two subsets of motor neurons ultimately express several phenotypic differences, including somal size, peripheral targets, and spinal cord locations. While somatic motor neurons remain ventrally, autonomic motor neurons (AMNs) move both dorsally and medially between embryonic days 14 and 18, when they approximate their final locations in spinal cord. The goal of the present investigation was to determine the potential guidance substrates available to AMNs during these movements. The dorsal translocation was studied in developing upper thoracic spinal cord, because, at this level, the majority of AMNs are located dorsolaterally. Sections were double-labeled by ChAT (choline acetyltransferase) and SNAP/TAG-1 (stage-specific neurite associated protein/transiently expressed axonal surface glycoprotein) immunocytochemistry to visualize motor neurons and the axons of early forming circumferential interneurons, respectively. Results showed that during the developmental stage when AMNs translocated dorsally, SNAP/TAG-1 immunoreactive lateral circumferential axons were physically located along the borders of the AMN region, as well as among its constituent cells. These findings indicate that lateral circumferential axons, as well as the SNAP/TAG-1 molecules contained upon their surfaces, are in the correct spatial and temporal position to serve as guidance substrates for AMNs. The medial translocation was studied in developing lower thoracic-upper lumbar spinal cord, because, at this level, more than half of the AMNs are medially located. Sections were double-labeled by ChAT and vimentin immunocytochemistry to visualize motor neurons and radial glial fibers, respectively. Observations on consecutive developmental days of the medial translocation revealed that AMNs were aligned with parallel arrays of radial glial fibers. Thus, the glial processes could serve as guides for the AMN medial movement. Future experimental analyses will examine whether circumferential axons and radial glial fibers are in fact functioning as migratory guides during AMN development, and, if so, whether specific surface molecules on these guides trigger the subsequent differentiation of AMNs.